ABSTRACT Rates of hepatocellular carcinoma (HCC) have been steadily increasing in the United States. One explanation for this observation is the alarming trend in obesity. A positive correlation between the development of non- alcoholic fatty liver disease (NAFLD) and HCC has been identified in the clinic, supporting a link between obesity and HCC. The public health impact of HCC is further exacerbated by the difficulty in detecting HCC tumors in the clinic, leading to the majority of tumors identified in the clinic presenting with preexisting metastasis. The aggressive nature of the disease at diagnosis results in a dismal five-year survival rate of 15 percent. The long-term goal of the proposed work is to delineate the longitudinal signaling perturbations that lead to NAFLD and subsequent progression to metastatic HCC. This project will: 1) develop a signaling profile of NAFLD, 2) delineate signaling changes associated with progression of HCC to a metastatic phenotype, and 3) investigate spatially localized signaling in metastatic HCC. As a central component of this work, a panel of direct protein kinase activity sensors will be employed for inflammatory, growth, survival, cell motility, and metabolic pathways. This panel of novel kinase activity sensors relies on the incorporation of a phosphorylation-sensitive amino acid into kinase-selective substrates, allowing for a direct fluorescence-based readout of kinase enzymatic activity in complex biological samples. To complement this initiative, this project will leverage a multidisciplinary approach to correlate observed changes in kinase signaling with changes in RNA and metabolite production. This multipronged approach will permit the identification of regulators, as well as effectors, of altered kinase activity observed in the disease state. Taken together, the proposed project will provide a quantitative picture of signaling perturbations in NAFLD, establish correlations between kinase activity and invasive phenotypes in HCC, and enable the analysis of spatially restricted kinase activity in HCC cell lines. In the long-term, this work will provide a basis for the development of clinical diagnostics and identify potential new targets for therapeutic intervention in the development and progression of HCC.